1. It is crucial during fixation, processing, and embedding in wax prior to sectioning, that all of the reagents completely penetrate and equilibrate within the embryonic tissue. Times vary according to the size of the embryo/tissue being prepared. The following illustrates modifications that have been used successfully on larger tissues (e.g., heads of E10 chick embryos and whole E18 mouse embryos).

On d 1, fix in 4% paraformaldhyde in 1X PBS overnight at 4°C.

On d 2, wash twice in PBS each for 30 min, wash twice in saline each for 30 min, and then incubate in 30% ethanol for 1 h, 50% ethanol for 1 h, 70% ethanol for 1 h, 100% ethanol for 3 h, and then in fresh 100% ethanol overnight.

On d 3, incubate in fresh 100% ethanol for a further 5 h. Then incubate in three changes of Histoclear (or xylene) the first for 1 h, the second for 3 h, and the last overnight.

On d 4, replace the Histoclear with freshly melted (52°C) wax, and incubate at 52°C in an oven for 3 h, replace the wax with fresh and incubate at 52°C for 5 h and then replace once more and incubate at 52°C overnight.

On d 5, incubate with two more changes of wax each for 3 h, and then embed the tissue.

2. Embryos can be stored at -20°C for up to a week at this stage.

3. Restriction enzyme sites should be selected to produce a transcript of 3001000 bp. If any larger, we find that nonspecific background signal increases. Also, if possible, select enzymes that produce a blunt end or a 5'-overhang, because 3'-overhanging ends facilitate "wraparound" transcripts resulting from the polymerase transcribing back along the complementary DNA strand. If there is no alternative to using an enzyme producing a 3'-overhanging end, the following procedure can be used after digestion to "end polish," generating a blunt end. After digestion, add Klenow polymerase to a final concentration of 5 U/|J.g DNA template, and incubate at 22°C for 15 min prior to proceeding with the rest of the transcription protocol.

4. Coplin jars containing 30-40 mL of solution are used at this stage and in Subheading 3.7., step 8 to reduce the volume of high-stringency buffer used, the large amounts of DTT required would otherwise prove very expensive.

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